Reaction mass of 604-916-7 and (4R,4aS,5aR,12aS)- 4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-9-nitro-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide sulfate (1:2),

Endpoint summary

Description of key information

Skin irritation:  

Two studies are available:  

1) An in vivo skin irritation study ( F.M. van Otterdijk, M.Sc., 2012) is available which is key study. This study showed that the test substance is not irritating to skin.  

2) An in vitro skin corrosion study ( Drs. C.A.F. Buskens 2011) is available which is key study. This study showed that the test substance is not corrosive to skin.

Eye irritation:

One study is availible:  

1) In vitro study is available (C.M. Verspeek-Rip, 2011) which is key study. This study showed that the test substance is corrosive or severe irritant in the bovine eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May 2011 to 13 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: Human Skin Model Test (adopted 13 April 2004).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Rationale
Recommended test system in international guidelines (OECD and EC).

Source
MatTek Corporation, Ashland MA, U.S.A.

Vehicle:

water

Details on test system:

Test system
EpiDerm Skin Model (EPI-200, Lot no.: 14763 kit N).
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg with 25 μl Milli-Q water was applied to each well plate

Duration of treatment / exposure:

3 minute, 1 hour

Number of replicates:

2 replicates per exposure time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

25

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

As the mean relative tissue viability for 9-Nitrominocycline was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment 9-Nitrominocycline is considered to be not corrosive.

Interpretation of results:

GHS criteria not met

Conclusions:

Finally, it is concluded that this test is valid and that 9-Nitrominocycline is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2011 to 03 June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No.404: "Acute Dermal Irritation/ Corrosion", Paris Cedex, 2002.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B4: "Acute Toxicity: Dermal Irritation/Corrosion". Official Journal of the European Union No. L142, May 2008, including most recent amendments.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.2500, Acute Dermal Irritation. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-98-196, August 1998.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 8147

Version / remarks:

Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 18.9 – 19.7ºC), a relative humidity of 40-70% (actual range: 31 - 68%) and 12 hours artificial fluorescent light and 12 hours darkness per day.

Accommodation
Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm). Acclimatization period was at least 5 days before start of treatment under laboratory conditions.

Diet
Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period

Water
Free access to tap water.

Results of analysis for diet (nutrients and contaminants) and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5 grams of the test substance, moistened with 0.4 mL of the vehicle,

Duration of treatment / exposure:

3 minutes (1 animal), 1 hour (2 animals)

Observation period:

4 hours

Number of animals:

3 Animals of 1 sex

Details on study design:

The study was performed in a stepwise manner and started with the treatment of one animal (sentinel) with a stepwise exposure regime. The two other animals were exposed to the substance for four hours only and in a similar manner 7 days later, after considering the degree of skin irritation observed in the first animal.

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

7.1. Irritation and corrosion (Table 1)
A 3-minute, 1-hour and 4-hour exposure to 0.5 g of 9-Nitrominocycline in the initially treated animal resulted in very slight erythema immediately after removal of the test substance. No skin irritation was observed among the other two animals following a 4-hour exposure period.

Other effects:

7.2. Coloration / Remnants
Yellow staining of the treated skin by the test substance was observed throughout the observation period which did not hamper the scoring of the skin reactions.
7.3. Toxicity / Mortality
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results 9-Nitrominocycline does not have to be classified and has no obligatory labelling requirement for skin irritation according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007),
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2011 to 12 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 437: "Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants" (adopted September 07, 2009).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants ". Official Journal of the European Union No. L324; Amended by EC No. 1152/2010 No. L142, 09 December 2010.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OTWG

Version / remarks:

The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX 127

Version / remarks:

In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Fundam Appl Toxicol 18:442-449

Version / remarks:

Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

other: an isolated bovine cornea

Details on test animals or tissues and environmental conditions:

Bovine eyes were used as soon as possible after slaughter on the same day.

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20% (w/w) test substance solution

Duration of treatment / exposure:

240 +/- 10 minutes

Observation period (in vivo):

Immediate opacity measurement and permeability evaluation of the cornea

Number of animals or in vitro replicates:

Three corneas for each treatment group (total 9 corneas)

Details on study design:

6.4.1. Preparation of corneas
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.

The isolated corneas were stored at 32 1 C in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 1 C. The corneas were incubated for the minimum of 1 hour at 32 1 C.

6.5. Cornea selection and Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 3 were not used. Three corneas were selected at random for each treatmentgroup.

6.6. Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 l of the negative control, 20% (w/v) Imidazole solution (positive control) or 20% (w/w) test substance solution were introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 10 minutes at 32 1 C. After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

6.7. Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.

The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

6.8. Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.

The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein /ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 5 minutes at 32 1 C.

6.9. Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 l of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (Multiskan spectrum, Thermo labsystems, Breda, The Netherlands). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

6.10. Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme: Multiskan spectrum version 1.00 (Thermo labsystems, Breda, The Netherlands) for optical density measurement.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

96

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: Permeability

Run / experiment:

Mean

Value:

0.144

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The negative control responses of the opacity and permeability values were less than the upper limits
of the laboratory historical range indicating that the negative control did not induce irritancy on the
corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 95 and
within the historical positive control data range (APPENDIX 3, Table 6). It was therefore concluded
that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

9-Nitrominocycline induced severe ocular irritation through one endpoint (opacity) mainly, resulting in a mean in vitro irritancy score of 98 after 240 minutes of treatment.
Since 9-Nitrominocycline induced an IVIS ≥ 55.1, it is concluded that 9-Nitrominocycline is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation:

Mean scores at 24, 48 and 72 hours for erythema were < 2.3 (actual value 0) and for edema were < 2.3 (actual value 0), therefore according to GHS criteria this substance was not considered a skin irritant.

 

Skin corrosion:

As the mean relative tissue viability for PF-06478031-01 was not below 50% (actual value 83%) after 3 minutes of treatment and not below 15% (actual value 25%) after 1 hour of treatment, it was not considered corrosive according to GHS criteria.

Serious eye damage/eye irritation:

9-Nitrominocycline induced severe ocular irritation through one endpoint (opacity) mainly, resulting in a mean in vitro irritancy score of 98 after 240 minutes of treatment. Since 9-Nitrominocycline induced an IVIS >= 55.1, it is concluded that 9 -Nitrominocycline is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability.